In this protocol, double stranded RNA is generated by simultanous synthesis of
sense and anti-sense transcripts. This avoids firstly the hybridisation of
individually synthesized transcripts and secondly precautions required for work
with mRNA, such as RNaseA traces, as dsRNA is not RNAseA sensitive.
Thirdly, dsRNA can be analysed by plain electrophoresis with agrose gels similar
to DNA.
1. Synthesis of the template
The promoter for T7 RNA polymerase is introduced by PCR. We use the following sequence
5' gta ata cga ctc act ata ggg cg + 15-20 nt specific for the target gene.
The length of the part that hybridizes to the target gene is chosen so that the
anneling temperature is about 55°C according to the formula
Tm = 4xGC + 2xTA - 5.
Genomic DNA or plasmid DNA (with the cDNA) are used as template. Correspondingly,
the PCR protocol is adjusted.
| 4 µl | Primer 1 |
| 4 µl | Primer 2 |
| 10 µl | Buffer (without Mg2+) |
| 2 µl | Template DNA |
| 69 µl | H2O |
| 2 µl | Taq polymerase (2 U) |
| 8 µl | Mg2+ |
| ----- | ------------------- |
| 80 µl | total volume |
|
PCR Program
| 95°C | 2 min |
| 95°C | 30 s |
| 55°C | 1 min |
| 72°C | 1 min 30 sec |
| repeat | 6 x |
| 95°C | 30 s |
| 60°C | 1 min |
| 72°C | 40 s |
| repeat | 30 X |
| 72°C | 5 min |
| 4°C | . |
|
The PCR product is either purified by agarose gel electrophoresis and excision of the
band followed by QiaQuick extraction or directly by PCR purification kit. The DNA
is eluted by 15-30
µl of DEPC-H20 or elution buffer. The yield should be about 5-7 µg of
DNA.
2. in vitro transcription by RiboMAX
| 20.25 µl | DEPC H2O |
| 4 µl | PCR product (about 1 µg) |
| 5 µl | 10X T7 Transcription Buffer Roche/Fermentas |
| 3.75 µl | CTP |
| 3.75 µl | ATP |
| 3.75 µl | UTP |
| 3.75 µl | GTP |
| 1.25 µl | RNAase inhibitor Roche |
| 2.5 µl | Pyrophosphatase |
| 2.0 µl | T7 RNA polymerase |
| ----- | ------------------- |
| 50 µl | total volume |
|
- Incubate for 4 hours at 37°C
- Add 1 µl of T7 RNA polymerase
- Incubate for 2 hours at 37°C
Final concentration of each NTP is 7.5 mM
3. Purification of dsRNA
- Add 3 µl DNaseI. Incubate 15 min in 37?
- Add DEPCH2O to 100 µl
- Add 100 µl phenol-chloroform-IAAÊ(25:24:1) to the same tube
- Mix 5 min
- Move the upper phase(~100 µl) to a new tube
- Add 100 µl chloroform and mix 5 min
- Move the upper phase(~100 µl) to a new tube
- Add 10 µl 3M NaAc (for RNA) and 2.5 fold 100% EtOH
- Mix 5 min
- -20°C overnight or -80°C 1 hour to precipitate the dsRNA
- Centrifuge at 4°C for 30 min
- Wash the pellet with cold 70% ethanol and centrifuge for another
5 min. Remove Ethanol
- After drying for 3 min, dissolve the pellet in 50 µl DEPC water.
- Use NanoDrop to measure the concentration. The yield should be more than 50 µg RNA.
The concentration should be in the range of 5 µg/µl.
- Analyse on a agarose gel to check the size.
4. Reagents, buffers
|